Reactive oxygen species (ROS) formed by the multi-component NADPH oxidase of phagocytic leukocytes play a primary role in host defense, but also perpetuate acute and chronic cellular inflammatory responses. Roles for ROS in non-phagocytic cells include intracellular signaling, proliferation, apoptosis, and innate immunity. We have shown that Rac2 serves as a key "molecular switch" for ROS formation in neutrophils. Our work suggested a novel 2-step mechanism through which Rac2 regulates the process of electron transfer from NADPH to molecular oxygen in the NADPH oxidase. A more detailed knowledge of NADPH oxidase regulation by Rac GTPase is critical for understanding the physiological and pathological regulation of ROS formation in both immune- and non-immune cells. Based upon predictions of our regulatory model, we will define the activity of Rac2 in controlling electron transfer reactions critical to phagocyte NADPH oxidase (phox) function. Interaction sites for Rac2 on cyt b and related cyt b (Nox) homologs will be defined. We will use a variety of experimental approaches to investigate Rac/Rho GTPase regulation of Nox function. Integrin-initiated signaling pathways that cross-talk with and regulate phox function, and their molecular targets in adherent neutrophils, will be identified. During cell activation, GDP Dissociation Inhibitors (GDIs) serve as key regulators of Rac activity. We recently identified RhoGDI phosphorylation as a mechanism for specific regulation of Rac GTPase release and activation. In the current application, we will characterize the regulation of GTPase-GDI interactions through tyrosine kinase-dependent phosphorylation of GDI. The biological roles of GDI kinases in modulating the GTPase-GDI regulatory cycle will be investigated.